Method of separation using aromatic thioether ligands

ABSTRACT

The present invention is a method of separating nucleic acid molecules from contaminants, such as proteins, in a solution and isolating one or more desired nucleic acid molecules, which method comprises the following steps (a) providing an aqueous adsorption solution, which includes nucleic acids and a salt that forms lyotropic ions when dissolved; (b) passing said solution over a matrix to adsorb the nucleic acids onto the matrix, said matrix including an aromatic ring moiety and at least one thioether moiety; (c) passing an aqueous eluent over said matrix to desorb the nucleic acid molecules therefrom, which eluent includes a salt that forms lyotropic ions and a gradient of increasing ionic strength originating from an increasing concentration of a salt that forms less lyotropic ions when dissolved than the ones present in said aqueous adsorption solution; and (d) isolating a fraction comprising the desired nucleic acid molecules.

TECHNICAL FIELD

The present invention relates to separation of nucleic acid molecules,especially plasmids, from other components in a solution. The methodutilises aromatic thioether ligands for adsorption of the nucleic acidmolecules and provides a novel scheme of desorbing the adsorbedmolecules, which greatly improves the separation efficiency of themethod all in all. The present method is preferably a chromatographicprocess.

BACKGROUND

In the last decade, the administration of therapeutic genes to patientshas become a reality for preventing or treating various diseases.Non-viral vectors are often preferred in clinical applications tominimise the risk of viral infections. This increases the demand forhighly purified plasmids for use in gene therapy and plasmid-basedvaccines. The stringent guidelines and rules set forth by healthauthorities require homogeneous preparations of purified supercoiledplasmid DNA for clinical applications.

Chromatography is the method of choice for both small- and large-scalepurification of supercoiled plasmid DNA (Ferreira, G. N. M., Prazeres,D. M. F., Cabral, J. M. S., and Schleef, M. Plasmid manufacturing—Anoverview. In: Schleef, M. (Ed.) Plasmids for therapy and vaccinationWiley-VCH: Weinheim, 2001, p. 193-236). Separation methods based onsize-exclusion, ion-exchange and hydrophobic interaction chromatography(HIC) have been shown to be suitable for purifying plasmid DNA(Ferreira, G. N. M., Monteiro, G. A., Prazeres, D. M. F., and Cabral, J.M. S. Downstream processing of plasmid DNA for gene therapy and DNAvaccine applications. TIBTECH 2000; 18, 380-388). Affinitychromatography, which is based on sequence-specific interactions betweenan immobilised synthetic oligonucleotide and a stretch of the plasmidDNA, has also been suggested (Schluep, T., and Cooney, C. L.Purification of plasmids by triplex affinity interaction. Nucleic AcidsResearch 1998; 26, 4524-4528). However, none of these techniques resultsin a homogeneous preparation of supercoiled plasmid DNA in sufficientquantities for non-analytical applications.

To circumvent this problem, efforts in the past were directed towardsthe development of sample preparation techniques that would minimise theformation of nicked forms of plasmid DNA. However, this approach wasdifficult to reproduce and the quality of the plasmid DNA thus produceddid not meet the stringent specifications set forth for the finalproduct (Levy, M. S., O'Kennedy, R. D., Ayazi-Shamlou, P., and Dunnill,P. Biochemical engineering approaches to the challenges of producingpure plasmid DNA. Trends in Biotechnology 2000; 18, 296-305).Accordingly, there has been a recognised need for developing a new androbust separation protocol for supercoiled plasmid DNA that wouldsignificantly reduce the tedious efforts for optimisation of theupstream processes.

Oscarsson et al. suggested thiophilic chromatography (Oscarsson, S., andPorath, J. Covalent chromatography and salt-promoted thiophilicadsorption. Analytical Biochemistry 1989; 176, 330-337; Porath, J.,Maisano, F., and Belew, M. Thiophilic adsorption—a new method forprotein fractionation. FEBS Letters 1985; 185, 306-310) to proteinpurification. In a patent application of later date, WO 95/33557,Oscarsson and Porath disclose an alkali-resistant protein adsorbent,which is similar to the ones discussed in 1989, but wherein thethiophilic group has been distanced from the rest of the ligand toimprove the properties in alkaline environments. Adsorption of proteinsis favoured by high concentrations of lyotropic salt, and desorption isachieved using the conditions conventionally used in hydrophobicinteraction chromatography (HIC), which is to replace the lyotropic saltby another, less lyotropic salt or simply by water.

More recently, the ability of different ligand structures comprisingthioethers to bind the different isoforms of plasmid DNA underconditions that would be applicable in large-scale processes have beeninvestigated. This was disclosed in Swedish patent application SE0101380.4, which however was not published at the time of the filing ofthe present application. Accordingly it was possible to narrow down thespecific structures that are required for an “optimal ligand” tointeract with supercoiled plasmid DNA and purify it selectively. As aresult, a new group of thiophilic ligands, namely aromatic thioethers,have been identified, which effectively differentiates between theisoforms of plasmid DNA. Media comprising these ligands have been shownto efficiently separate supercoiled (covalently closed circular (ccc))plasmid DNA from its isoform, i.e. open circular (oc) form in a singlechromatography step. Accordingly, the use of these media appears to bepromising and should facilitate the production of highly purifiedsupercoiled plasmid DNA for use in gene therapy and DNA vaccineapplications.

However, even though plasmid DNA can be separated from its isoform,other problems remain to be solved. For example, a cell lysatecomprising a desired nucleic acid will usually also comprise one or moreproteins, such as enzymes, or various degradation products. Even thoughpreviously reported methods in principle are capable of separatingprotein from desired nucleic acids, the resolution obtained is still notfully satisfactory for an efficient production process. Cell lysatesthat comprise deoxynucleic acid (DNA) will normally also compriseribonucleic acid (RNA) and some remaining proteins. However, in theabove discussed context when plasmid DNA is desired for pharmaceuticalpurposes, both will constitute contaminants that has to be removed.Furthermore, chromatographic purification of plasmid DNA results in mostcases in co-purification of endotoxins, i.e. membrane components of hostbacteria, that have to be removed to fulfil the rigorous requirementsfor pharmaceutical products. Accordingly, there is a need ofimprovements of the presented purification protocols using thiophilicchromatography as regards the separation of nucleic acids from theabove-discussed contaminants.

SUMMARY OF THE PRESENT INVENTION

One object of the present invention is to provide an alternative methodof separating nucleic acids, such as DNA, from a solution using a matrixcomprising one or more aromatic thioether ligands. This and otherobjects can be achieved by the method disclosed in the appended claims.

A more specific object of the present invention is to provide a methodof desorbing molecules bound to such matrices, which method provides amore efficient separation of the plasmids from one or more undesiredcomponents, such as proteins, RNA, and/or endotoxins.

Another object of the invention is to provide a method of desorbing asmentioned above, which also is capable of separating different plasmidisoforms from each other, more specifically ccc plasmid DNA from ocplasmid DNA.

Definitions

The term “proteineous” as used herein includes whole protein as well asparts or traces of protein and other molecules comprising a peptidicstructure.

The term “nucleic acid molecules” is used herein synonymously with theterm “nucleotides” and includes DNA, e.g. plasmids, such as opencircular (oc or nicked) plasmid DNA, supercoiled (ccc) plasmid DNA andother DNA, such as-genomic. DNA, as well as RNA, such as mRNA, tRNA andsRNA.

The phrase to “pass a solution/eluent over a matrix” means any way ofcontacting a matrix with a solution or eluent as well as removal thereofafter a certain period of time. Accordingly, the term includes dynamicchromatographic methods as well as batch procedures.

The term “eluent” is used herein in its conventional meaning inchromatography, i.e. a solution capable of perturbing the interactionbetween the solid phase (adsorbent matrix) and product (nucleic acidmolecules) and promoting selective dissociation of the product from thesolid phase.

The term “lyotropic” is a measure of the ability of ions to influencethe hydrophobic character of the interactions in a solvent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows examples of aromatic thioether ligands, which possessseparation properties and hence are useful according to the invention.

FIG. 2 shows a chromatogram obtained after group separation of alkalinelysate, resulting in separate plasmid DNA and RNA fractions.

FIG. 3 a-d shows results obtained after chromatography on the ligandsdepicted in the inserts. FIG. 3 e shows electropherograms after laserinduced fluorescence capillary gel electrophoresis of samples obtainedafter chromatography on aromatic thioether ligands.

FIG. 4 is a chromatogram illustrating binding of clarified alkalinelysate on an aromatic thioether ligand and elution of the differentnucleic acid fractions.

FIG. 5 is a chromatogram depicting the profile of plasmid DNA, RNA andlipopolysaccharides after chromatography on an aromatic thioetherligand.

FIG. 6 shows the result obtained after chromatography of immunoglobulinson an aromatic thioether ligand.

FIG. 7 is a chromatogram illustrating elution of plasmid DNA by anincreasing KCl gradient

FIG. 8 is a chromatogram demonstrating the use of Na₂SO₄ as lyotropicsalt for the purification of plasmid DNA on aromatic thioether ligands.

FIG. 9 shows chromatograms illustrating the difference in endotoxinprofile when comparing decreasing salt elution (FIG. 9 a) withincreasing salt elution (FIG. 9 b).

DETAILED DESCRIPTION OF THE PRESENT INVENTION

More specifically, a first aspect of the present invention is a methodof separating nucleic acid molecules from contaminants in a solution,which method comprises the following steps

-   (a) providing an aqueous adsorption solution, which comprises    nucleic acids and a salt that forms lyotropic ions when dissolved;-   (b) passing said solution over a matrix to adsorb the desired    nucleic acids onto the matrix, which comprises an aromatic ring    moiety and at least one thioether moiety;-   (c) optionally washing the matrix;-   (d) passing an aqueous eluent over said matrix to desorb the nucleic    acid molecules therefrom, which eluent in addition to a salt that    forms lyotropic ions also comprises a gradient of increasing ionic    strength originating from an increasing concentration of salt that    forms less lyotropic ions when dissolved than the ones present    during adsorption; and-   (e) isolating the fraction comprising the desired nucleic acid    molecules.

In the most preferred embodiment, the ions referred to in steps (a) and(d) are the anions formed as each respective salt is dissolved.

The method according to the invention can be used to isolate nucleicacid molecules expressed in cells, for example in recombinant cells.Consequently, it also comprises a first step of disintegrating the cellsto provide the solution comprising nucleic acid molecules. Suchdisintegration is performed e.g. by lysis, such as alkaline lysis,according to standard protocols (see e.g. Maniatis, T, Fritsch, E. F.and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press, Cold Spring Harbour, N.Y.).Accordingly, the solution provided in step (a) above is preferablyessentially free of cell debris. In addition, the present invention isalso useful for analysis of nucleotide mixtures in solution, i.e. aseparation for analytical purposes.

In an advantageous embodiment of the present method, the fractionisolated according to step (e) is plasmid DNA, which is essentially freefrom RNA. As discussed above, this is a strict requirement inpharmaceutical preparations. As will be shown in the experimental partbelow, the RNA maintains firmly bound to the matrix during the elutionof DNA. If desired, the RNA can then easily be removed by washing thematrix with water or low salt buffer, e.g. for regeneration of thematrix. However, in an alternative embodiment, the present invention isa method whereby RNA is recovered, e.g. for scientific research, from asolution that also comprises DNA. Both these embodiments exhibit theclear and unexpected advantage of separating nucleic acid molecules notonly from each other, but also from contaminants, such as proteineouscomponents and endotoxins, present in a solution. As will be shown inthe experimental part, protein that binds to the matrix used accordingto the present invention are eluted at a different stage than thenucleic acids, and can accordingly be collected in a separate fraction.

The present invention shows that it is possible to separate bothproteineous components and RNA from the DNA in a cell lysate by use of anovel principle of elution from matrices comprising aromatic thioetherligands. In addition, the present method has also been shown toefficiently separate a desired nucleic acid molecule from othercontaminants, such as endotoxins. Accordingly, the method according tothe invention is useful e.g. for purification of nucleic acids for usein gene therapy, DNA vaccines and laboratory studies related to genetherapy. In an advantageous embodiment, the present method will provideisolated supercoiled plasmid DNA of acceptable gene therapy grade. Asmentioned above, the previously described methods for isolation of suchcarriers have not been satisfactory to this end.

In one embodiment of the present method, the solution provided in step(a) comprises a dissolved alkali salt, such as ammonium sulphate orsodium sulphate. Accordingly, the anions present therein during theadsorption will be the strongly lyotropic sulphate ions. In a specificembodiment, the concentration of said salt is below about 3.0 M, such asabout 2.5 M. As the skilled person in this field will realise, the upperlimit is determined by the solubility of each salt. However, as theskilled in this field will realise, different salt concentrations may berequired in different situations, not only depending on the specificligand used on the matrix, but also on the nucleic acid to be separated.The pH of the solution during adsorption step can differ from the oneduring the elution step. However, to assure the stability of the nucleicacids to be separated, the pH should remain at physiological pH, i.e. inthe range of about 6.5-8.5.

In an advantageous embodiment, the matrix to which the desired nucleicacids have been adsorbed is washed before elution according to step (d).During washing, loosely bound contaminants are removed. Such washing caneasily be performed by the skilled in this field according to standardprocedures.

Step (d) according to the present invention is performed with a suitableaqueous eluent, which in addition to a salt that forms lyotropic ionsalso comprises a gradient of increasing ionic strength, for example acontinuous gradient. However, for certain purposes as large-scaleproduction of supercoiled plasmid DNA, it may be desired to use astep-wise gradient.

The increasing ionic strength of the eluent used in step (d) is providedby an increasing concentration of a salt in the eluent, which salt iscapable of forming less lyotropic ions, such as anions, than the onedissolved in the adsorption solution. As is well known to the skilled inthis field, the lyotropic properties of ions can be rated according tothe Hofmeister or lyotropic series. The dissolved salt will graduallychange the conditions in the solvent, which change was found by thepresent inventor to be useful to desorb DNA, and RNA as easily separatedfractions. In fact it was found that for most ligands, RNA is eluted asthe last fraction simply by addition of water. Proteins and endotoxinscan only be removed by the addition of water, indicating a solelyhydrophobic binding mechanism of these molecules to the matrix,completely different from the mechanisms nucleic acids deploy. In oneembodiment, the salt dissolved in the eluent is an alkali salt, such assodium or potassium chloride. Thus, the less lyotropic anions arechloride ions. In a specific embodiment, the maximum concentration ofthe salt is about 3 M. However, as mentioned above in relation to thesalt dissolved in the adsorption solution, for each specific ligand anddesired nucleic acid, some routine testing may be needed in order todetermine optimal conditions. Similar to the adsorption step, the pHvalue during elution should be kept in the range where the nucleic acidsare stable.

Thus, the present invention is based on the unexpected finding, thatnucleic acids adsorbed to a matrix via aromatic thioether ligands canefficiently be eluted by a gradient of increasing ionic strength asdiscussed above. More specifically, the eluent is comprised of a saltthat forms lyotropic ions, such as the buffer used in the adsorptionstep, and in addition to that a gradient of increasing ionic strength.This is quite contrary to the recently introduced use of aromaticthioether ligand matrices, such as in the above-discussed WO 95/33557,wherein elution was performed by removing the lyotropic salt from theelution buffer. Thus, in the prior ark, elution of aromatic thioetherhas been performed either by adding an aqueous solution, which dilutesthe lyotropic salt, or by adding a salt that replaces the lyotropicsalt. In other words, the total salt concentration has been reduced orkept at a similar value during elution. However, the present inventor isthe first to show that elution of aromatic thioether ligands can beperformed by use of the same buffer as used in the adsorptionsupplemented with a gradient of increasing ionic strength.

As regards the matrix used in the present method, it is comprised of aligand as discussed in more detail below coupled to a support via athioether bond. Each matrix may comprise one or more ligand structures.

In one embodiment, the ligand used comprises an aromatic thioetherwherein the aryl group is selected from pyridyl, phenyl, benzyl, toluyl,phenethyl, naphtyl, imidazolyl, pyrazolyl, pyrazinyl, pyrimidinylpyridazinyl, piperidinyl, morpholinyl, piperazinyl, indolyl quinolinyland purinyl groups.

In an advantageous embodiment, the pyridyl-S and/or phenyl groupcomprises further substituents on the aryl group, for example asillustrated in FIG. 1.

The ligand attached to the carrier material to form the matrix of theinvention can be a mercapto-pyridine, mercaptoalkylpyridine. Hereby itis the mercapto-group that is attached to the carrier material via athioether binding. Further, as mentioned above, aryl groups forming partof the ligand can be phenyl, benzyl toluyl, phenethyl, naphtyl,imidazolyl, pyrazolyl, pyrazinyl pyrimidinyl, pyridazinyl, piperidinyl,morpholinyl piperazinyl, indolyl, quinolinyl purinyl. Furthersubstituents can also be added on the aromatic ring. By providing thepresent aromatic thioether ligands with additional substituents, a largerange of different separation media can be designed. The substituentscan for example be one or more amine groups, nitro groups, ether groups,thiol groups and/or halogens, such as fluorine. These additionalsubstituents can also comprise further carbon atoms, as desired. Also,as the skilled in this field will realise, carbon atoms can be exchangedfor heteroatoms in the above discussed ring structures. It is to beunderstood herein that the term “aromatic thioether ligand” comprises alarge range of compounds that can be substituted to a desired extent,some of which will be exemplified below in FIG. 1. The ligand densitycan be 10-500 μmole/mL carrier, preferably in the range 10-100 μmole/mLcarrier.

The matrix used in the present invention comprises ligands that may beof one the same or different chemical structures, coupled to a support.The support can be in any suitable form, such as essentially sphericalparticles, membranes, filters, microtiter plates etc. The support can beany suitable, well-known inorganic or organic material. Examples ofinorganic support materials are glass, silica, or other inertparticulate minerals. Examples of organic support materials are agarose,dextran, divinylbenzene etc. There are many commercial productsavailable based on different resins or polymer, e.g. agarose orcross-linked agarose (such as Sepharose™, Amersham Biosciences, Uppsala,Sweden), dextran (such as Sephadex™, Amersham Biosciences, Uppsala,Sweden), polystyrene/divinylbenzene (MonoBeads™, SOURCE™, AmershamBiosciences, Uppsala, Sweden), coated polystyrene, acrylic polymer,dextran acrylic polymer (Sephacryl™, Amersham Biosciences, Uppsala,Sweden), vinylic grafted polymer, or vinylic polymer, different silicabased resins such as silica-dextran, silica-acrylic polymer andsilica-polyethyleneimine. In an advantageous embodiment, the matrix iscross-linked agarose, which for example may have been functionalisedwith ligands using epoxy-activation.

The present process may be performed with the matrix arranged in theform of an expanded bed or a packed bed, and can be dynamic, i.e.chromatography, or run in a batch mode. In packed bed adsorption, thematrix is packed in a chromatographic column and all solutions usedduring a purification process are passed through the column, usually inthe same direction. In expanded bed adsorption however, the matrix isexpanded and equilibrated by applying a liquid flow through the column,usually from beneath. A stable fluidised expanded bed is formed whenthere is a balance between particle sedimentation or rising velocity andthe flow velocity during application of the sample and washing steps. Inthe elution step of an expanded bed, the matrix is precipitated andbehaves like a packed bed matrix.

In one embodiment, the matrix particles are of a mean size in the rangeof about 10-300 μm, e.g. within a range of 10-20, 20-50, 50-100, 100-200or 200-300 μm. However, the particles can advantageously be prepared inany size for which commercially available sieve equipment is available,such as 250, 212, 180, 150, 125, 106, 90, 75, 63, 45, 37, 30, 25, 20, 15μm.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows examples of a variety of aromatic thioether compoundsreacted with Sepharose™ 6 Fast Flow (Amersham Biosciences, Uppsala,Sweden). As appears from this drawing, the aromatic thioether compoundscan be provided with different substituents, each of which can result inbinding and elution properties that may prove advantageous for differentpurposes.

FIG. 2 illustrates preparation of plasmid DNA. Alkaline lysate is loadedon a Sepharose™ 6 Fast Flow (Amersham Biosciences, Uppsala, Sweden)column preconditioned in 2.0 M (NH₄)₂SO₄, 10 mM EDTA, 100 mM Tris-HCl pH7.0. After chromatography at 30 cm/h, plasmid DNA is separated from RNA.Furthermore, plasmid DNA can now be collected in the buffer required forfurther experiments with thiophilic aromatic chromatography.

FIGS. 3 a and 3 b illustrate the purification of supercoiled plasmid DNAon different columns with aromatic thioether ligands according to theinvention. The plasmid DNA sample is prepared as in FIG. 2 and loaded ona column preconditioned in 2.0 M (NH₄)₂SO₄, 10 mM EDTA, 100 mM Tris-HCl,pH 7.0. Desorption of the supercoiled plasmid DNA is obtained with agradient to 2M NaCl in the same buffer. Under these conditions, opencircular plasmid DNA does not bind to the matrix, while supercoiledplasmid DNA binds and can be eluted by increasing NaCl-concentration.FIGS. 3 c and 3 d are comparative examples of similar ligands, thathowever, are either not aromatic (FIG. 3 c) or have no thioether present(FIG. 3 d). The experiments are run with the same sample and buffers asin FIGS. 3 a and 3 b. It is clear from the chromatograms that none ofthese last-mentioned ligands are suited for the type of chromatographydescribed in FIG. 3 a and FIG. 3 b. FIG. 3 e depicts electropherogramsafter laser induced fluorescence capillary gel electrophoresis ofsamples obtained in experiments analogous to FIG. 3 a. Open circularform of plasmid DNA was not retained by the aromatic thioether ligand,while supercoiled forms of the plasmid were adsorbed and eluted early inthe linear gradient.

FIG. 4 illustrates adsorption and desorption of alkaline lysate to athiophilic aromatic-chromatography matrix. The clarified alkaline lysatesample is diluted with 4M M (NH₄)₂SO₄, 10 mM EDTA, 100 mM Tris-HCl, pH7.0 to obtain a final concentration of 2.25M (NH₄SO₄. In 2.25 M(NH₄)₂SO₄, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0, all nucleic acidmolecules adsorb to the matrix. Desorption is however different for thedifferent nucleic acids. Isoforms of the plasmid DNA can be separated bya gradient to 3 M NaCl in the 2.25 M (NH₂)₂SO₄, 10 mM EDTA, 100 mMTris-HCL pH 7.0 buffer (insert at higher magnification), while RNA canonly be eluted from the matrix with a gradient to less (NH₄)₂SO₄,resulting in a complete and robust separation of the different types ofnucleic acid molecules.

FIG. 5 depicts the separation between plasmid DNA andlipopolysaccharides (LPS). Plasmid DNA is spiked with RNA andfluorescent LPS and loaded on 2-mercaptopyridine Sepharose™ 6 Fast Flow(Amersham Biosciences, Uppsala, Sweden) equilibrated in 2.25 M(NH₄)₂SO₄, 10 mM EDTA, 100 mM Tris-HCL pH 7.0. Plasmid DNA isoformselute by increasing the conductivity in a gradient to 3M NaCl inequilibration buffer, while RNA and LPS elute by decreasing theconductivity by a gradient to water.

FIG. 6 shows how immunoglobulins can adsorb to 2-mercaptopyridinSepharose™ 6 Fast Flow (Amersham Biosciences, Uppsala, Sweden) in 1.0 M(NH₄)₂SO₄, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0. They can however not beeluted by increasing the conductivity with NaCl. For desorbing theproteins, conductivity has to be decreased by lowering the(NH₄)₂SO₄-concentration. This indicates a hydrophobic binding principle,different from the binding mechanism for nucleic acid molecules to thearomatic thioether-type ligand used according to the invention.

FIG. 7 shows that other salts for eluting the plasmid DNA molecules canreplace NaCl. Plasmid DNA, prepared as in FIG. 2 is loaded on2-mercaptopyridin Sepharose™ 6 Fast Flow (Amersham Biosciences, Uppsala,Sweden) in 2.0 M (NH₄)₂SO₄, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0 andeluted with a gradient to 1M KCl in loading buffer.

FIG. 8 illustrates how plasmid DNA is loaded on a column preconditionedin 3.0 M Na₂SO₄, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0 and eluted with agradient to 1M NaCl in the buffer used for preconditioning. Thus, thefigure demonstrates the replacement of the hydrophobic salt used forbinding of nucleic acid molecules to the thiophilic aromaticchromatography matrix.

FIGS. 9 a and b illustrate the differences in endotoxin profile wheneluting from an aromatic thioether ligand used according to theinvention with either decreasing or increasing salt concentration.Plasmid DNA is prepared as in FIG. 2 and loaded on 2-mercaptopyridinSepharose™ 6 Fast Flow (Amersham Biosciences, Uppsala, Sweden) at 2.0 M(NH₄)₂SO₄, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0. Consequently supercoiledplasmid DNA is eluted with either a gradient to water (FIG. 9 a) or agradient to 2M NaCl, 2.0 M (NH₄)₂SO₄, 10 mM EDTA, 100 mM Tris-HCl, pH7.0 followed with a gradient to water (FIG. 9 b). Some fractions weretested for endotoxin content using the standard LAL-test. From theexperiment it is clear that the major bulk of endotoxins elute whendecreasing the conductivity. Consequently, supercoiled plasmid DNA ispreferably eluted by increasing the conductivity. In this manner, thesupercoiled plasmid DNA is eluted and can be collected withoutco-elution of endotoxins.

Experimental Part

Below the present invention will be disclosed by way of examples, whichare intended solely for illustrative purposes and should not beconstrued as limiting the present invention as defined in the appendedclaims. All references mentioned below or elsewhere in the presentapplication are hereby included by reference.

Chromatograph Media Prototypes

All ligands (inserts in FIG. 2 a to 2 d) were coupled to epoxy-activatedSepharose™ 6 Fast Flow (Amersham Biosciences, Uppsala, Sweden) byestablished methods (Hermanson, G. T., Mallia, A. K. & Smith, P. K.Immobilised affinity ligand techniques. Academic Press Limited, London,1992, p. 51-132). The ligand concentration was chosen to be in excessover available binding sites and after washing, the amount of coupledligand was checked by elemental analysis (sulphur or nitrogen).

Plasmid DNA

A 6125 base pair plasmid composed of pUC19 plasmid with an insertrepresenting part of protein A (the identity of the insert is notimportant here) was used for this study. The plasmid was transfected andgrown in E. coli TG1α by well-established protocols (Sambrook, J., andRussel, D. W. Molecular cloning: a laboratory manual, Cold SpringHarbor, N.Y., 2001). Clarified alkaline lysate was prepared according toHorn et al. (Horn, N. A, Meek, J. A., Budahazi, G., and Marquet, M.Cancer gene therapy using plasmid DNA: purification of DNA for humanclinical trials. Human Gene Therapy 1995; 6, 565-573). A crude plasmidDNA preparation (containing both open circular and supercoiled isoforms)was prepared and used in FIG. 2. 1 litre of clarified alkaline lysate,was loaded on Sepharose™ 6 Fast Flow (Amersham Biosciences, Uppsala,Sweden) packed in an INdEX™ 100 column (Amersham Biosciences) at 30 cm/hin order to change the buffer to the desired SO₄ ²⁻-concentration in 10mM EDTA, 100 mM Tris-HCl, pH 7.0 (2.0M (NH₄)₂SO₄ in FIGS. 3, 7 and 9;2.25M (NH₄)₂SO₄ in FIGS. 4 and 6; 3.0M Na₂SO₄ in FIG. 8).Simultaneously, this procedure also removed RNA and other contaminants(not shown).

Chromatographic Procedures

All column chromatography was performed with an ÄKTA™explorer (AmershamBiosciences) at 75 cm/h. Samples were loaded on columns preconditionedin the appropriate SO₄ ²⁻-concentration in 10 mM EDTA, 100 mM Tris-HCl,pH 7.0 (1.0M (NH₄)₂SO₄ in FIG. 5; 2.0M (NH₄)₂SO₄ in FIGS. 3, 7 and 9;2.25M (NH₄)₂SO₄ in FIGS. 4 and 6; 3.0M Na₂SO₄ in FIG. 8) and eluted witha gradient to salt in the same buffer (2M NaCl in FIG. 3 a-d, 3M NaCl inFIGS. 4, 5, 6, 8 and 9 b, 1M KCl in FIG. 7) or to water (FIG. 9 a) in 10(FIGS. 3, 5 and 6) or 5 (FIGS. 4, 7, 8 and 9 a-b) column volumes. InFIGS. 4, 5, 6, 8 and 9 b, the salt gradient is followed by a watergradient for complete removal of contaminants.

Fluorescent-Lipopolysaccharides

To estimate the elution profile of endotoxins in the chromatographicmethod, five ml of a sample containing the different plasmid DNAisoforms was spiked with RNA and 2.5 μg/ml of a fluorescently labelled10.000 Da lipopolysaccharide isoform from E. coli (Isotype 055:B5,Molecular Probes, Leiden, The Netherlands). Fractions were collected andfluorescence was recorded according to the manufacturers' instructions.

Analytical Procedures

Peaks 1, 2, 3 and 4 in FIGS. 4 and 5 were judged to be non-nucleic acid,open circular plasmid DNA, supercoiled plasmid DNA and RNA respectivelyby their 260 nm/280 nm absorption ratios and their electrophoreticmobility pattern on 1% agarose gel electrophoresis and ethidium bromidestaining (Sambrook, J., and Russel, D. W. Molecular cloning: alaboratory manual, Cold Spring Harbor, N.Y., 2001) (not shown).

Endotoxin values were determined with the standard LAL-test according tothe manufacturer's instructions.

Laser induced fluorescence capillary gel electrophoresis was performedas described by Schmidt et al. (Schmidt, T., Friehs, K, and Flaschel, E.(2001): Structures of plasmid DNA, in: M. Schleef (Ed.): “Plasmids fortherapy and vaccination”, pp.29-43, Wiley-VCH, Weinheim).

1. A method of separating nucleic acid molecules from contaminants in asolution and isolating one or more desired nucleic acid molecules, whichmethod comprises the following steps (a) providing an aqueous adsorptionsolution, which includes nucleic acids and a salt that forms lyotropicions when dissolved; (b) passing said solution over a matrix to adsorbthe nucleic acids onto the matrix, said matrix including an aromaticring moiety and at least one thioether moiety; (c) passing an aqueouseluent over said matrix to desorb the nucleic acid molecules therefrom,which eluent includes a salt that forms lyotropic ions and a gradient ofincreasing ionic strength originating from an increasing concentrationof a salt that forms less lyotropic ions when dissolved than the onespresent in said aqueous adsorption solution; and (d) isolating afraction comprising the desired nucleic acid molecules.
 2. The method ofclaim 1, wherein the fraction isolated is plasmid DNA, which isessentially free from proteineous components and/or RNA and/orendotoxins.
 3. The method of claim 1, wherein the lyotropic and lesslyotropic ions of steps (a) and (c) are anions formed of the respectivesalts.
 4. The method of claim 1, wherein the salt in step (a) isammonium sulphate or sodium sulphate.
 5. The method of claim 4, whereinthe salt in the adsorption solution is at a concentration below about 3M.
 6. The method of claim 1, wherein the salt dissolved in the eluent isan alkali salt, such as sodium chloride or potassium chloride.
 7. Themethod of claim 6, wherein the salt in the eluent is at a concentrationno greater than about 3 M.
 8. The method of claim 1, wherein the matrixis comprised of a carrier to which an aromatic thioether ligand has beencoupled, wherein in said ligand the aryl group is selected from thegroup consisting of pyridyl, phenyl, benzyl, toluyl, phenethyl, naphtyl,imidazolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, piperidinyl,morpholinyl, piperazinyl, indolyl, quinolinyl, and purinyl groups. 9.The method of claim 8, wherein the ligand has been coupled to thecarrier via the thioether moiety.
 10. The method of claim 1, furthercomprising the step of washing said matrix after step (b).